Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

Summary

The goal of this protocol is to test the ability of progenitor cells derived from human perivascular adipose tissue to differentiate into multiple cell lineages. Differentiation was compared to mesenchymal stem cells derived from human bone marrow, which is known to differentiate into adipocyte, osteocyte, and chondrocyte lineages.

Abstract

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages. Adipogenic differentiation of progenitor cells is a major mechanism driving adipose tissue expansion and dysfunction in response to obesity. Understanding changes to perivascular adipose tissue (PVAT) is thus clinically relevant in metabolic disease. However, previous studies have been predominately performed in the mouse and other animal models. This protocol uses human thoracic PVAT samples collected from patients undergoing coronary artery bypass graft surgery. Adipose tissue from the ascending aorta was collected and used for explantation of the stromal vascular fraction. We previously confirmed the presence of adipose progenitor cells in human PVAT with the capacity to differentiate into lipid-containing adipocytes. In this study, we further analyzed the differentiation potential of cells from the stromal vascular fraction, presumably containing multi-potent progenitor cells. We compared PVAT-derived cells to human bone marrow MSC for differentiation into adipogenic, osteogenic, and chondrogenic lineages. Following 14 days of differentiation, specific stains were utilized to detect lipid accumulation in adipocytes (Oil red O), calcific deposits in osteogenic cells (Alizarin Red), or glycosaminoglycans and collagen in chondrogenic cells (Masson’s Trichrome). While bone marrow MSC efficiently differentiated into all three lineages, PVAT-derived cells had adipogenic and chondrogenic potential, but lacked robust osteogenic potential.

Introduction

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic lineages¹. This tissue expands through hypertrophy of mature adipocytes and de novo differentiation of resident MSC to adipocytes. Perivascular adipose tissue (PVAT) surrounds blood vessels and regulates vascular function^2,3. Obesity-induced PVAT expansion exacerbates cardiovascular pathologies. While the multipotent potential of MSC from human subcutaneous adipose depots have been well studied^4,5, no studies have explanted and evaluated the differentiation capacity of human PVAT-derived progenitor cells, likely due to the invasiveness of procurement. Thus, the goal of this work is to provide a methodology to explant and propagate progenitor cells from human aortic PVAT from patients with cardiovascular disease and to test their propensity to differentiate to osteogenic, chondrogenic, and adipogenic lineages. Our source of PVAT is from the site of anastomosis of the bypass graft on ascending aorta of obese patients undergoing coronary artery bypass graft surgery. Freshly-isolated PVAT is enzymatically-dissociated and the stromal vascular fraction is isolated and propagated in vitro, enabling us to test for the first time the differentiation capacity of human PVAT-derived progenitor cells.

Using primary cultured human PVAT stromal vascular fraction, we tested three assays designed to induce stem/progenitor cells to differentiate toward adipogenic, osteogenic, or chondrogenic lineages. Our prior study identified a population of CD73+, CD105+, and PDGFRa+ (CD140a) cells that can robustly differentiate into adipocytes⁶, although their multipotency was not tested. PVAT directly regulates vascular tone and inflammation⁷. The rationale for testing the differentiation potential of this novel cell population is to begin to understand the specialized influence of PVAT on vascular function, and mechanisms of PVAT expansion during obesity. This methodology enhances our understanding of the functions of adipose-tissue derived progenitor cells and enables us to identify and compare similarities and differences of progenitor cells from different tissue sources. We build upon established and validated approaches for isolating and differentiating MSC towards different lineages and optimize procedures to maximize the viability of human PVAT-derived progenitor cells. These techniques have broad applications in the fields of stem and progenitor cell research and adipose tissue development.

Protocol

The use of human tissues in this study was evaluated and approved by the Institutional Review Board of Maine Medical Center, and all personnel received appropriate training prior to experimentation.

1. Preparations

1. Make dissociation buffer by reconstituting 50 mg animal-free collagenase/dispase blend I solution with 1 mL of nanopure H₂O. Prepare 1 mg/mL working solution by adding 49 mL of high glucose DMEM containing 1% w/v BSA to the reconstituted collagenase/dispase solution. Store 5 mL working aliquots at -20 °C and warm to 37 °C using a water bath prior to use.
2. Make antibiotic solution by adding 1 mL of 100x antibiotic/antimycotic solution (final concentration is 200 units/mL penicillin, 200 units/mL streptomycin and 0.5 µg/mL fungizone) to 49 mL of HBSS, and store on ice.
3. Prepare gelatin solution (0.2% w/v) by dissolving 200 mg gelatin from bovine skin in 100 mL of nanopure H₂O. Autoclave the solution for 20 min and store at 4 °C.
4. Prepare 500 mL of PVAT growth media: high glucose DMEM/F12 with glutamine supplement, sodium pyruvate, and sodium bicarbonate, supplemented with 10% FBS and 100 µg/mL antimicrobial agent (see Table of Materials).
5. Prepare 50 mL adipogenic induction media: 40.8 mL high glucose DMEM, supplemented with 7.5 mL PVAT growth media, 500 µL of 100x antibiotic/antimycotic solution (final concentration is 100 units/mL penicillin, 100 units/mL streptomycin and 0.25 µg/mL fungizone), 0.5 mM IBMX (500 mM stock in 0.1 M NaOH), 1 µM dexamethasone (1 mM stock in ethanol), 5 µM rosiglitazone (5 mM stock in DMSO), 33 µM biotin (66 mM stock in DMSO), 100 nM insulin (200 µM stock in 0.1% acetic acid), and 20 µM pantothenic acid (100 mM stock in H₂O).
6. Prepare 50 mL of control induction media for the adipogenic lineage: 40.8 mL high glucose DMEM supplemented with 7.5 mL PVAT growth media and 500 µL of pen-strep (100x stock).
7. Prepare 50 mL adipogenic maintenance media: 41.5 mL high glucose DMEM supplemented with 7.5 mL PVAT growth media from step 1.3, 500 µL pen-strep (100x stock), 1 µM dexamethasone (1 mM stock in EtOH), 33 µM biotin (66 mM stock in DMSO), 100 nM insulin (200 µM stock in 0.1% acetic acid), and 20 µM pantothenic acid (100 mM stock in H₂O).
8. Prepare 500 mL of growth media for the osteogenic and chondrogenic lineages: high glucose αMEM supplemented with 10% FBS, 1x glutamine supplement (see Table of Materials), and 5 mL of pen-strep (100x stock).
9. Prepare 50 mL of induction media for the osteogenic lineage: 50 mL of the bone marrow MSC growth media supplemented with 10 nM dexamethasone and 10 mM β-glycerophosphate.
10. Prepare 50 mL of induction media for the chondrogenic lineage: 50 mL of the bone marrow MSC growth media supplemented with 100 nM dexamethasone, 50 µg/mL sodium ascorbate-2-phosphate, and 10 ng/mL TGFβ1. For the osteogenic and chondrogenic conditions, the basal bone marrow MSC growth media will serve as the non-induction media.

2. Protocol 1: Culture Human PVAT Cells from the Stromal Vascular Fraction

NOTE: PVAT is resected from the site of graft anastomosis on the ascending aorta of anesthetized patients undergoing coronary artery bypass graft procedures. Aortic PVAT is placed in a 15 mL conical containing 10 mL ice cold high glucose DMEM F12 and transferred from the operating room to the laboratory within 2 h of resection. Aortic PVAT is discarded tissue during bypass procedure and has been deemed as non-human subjects research by Maine Medical Center’s Internal Review Board.

1. This protocol is for a ~500 mg piece of human PVAT (approximately 3 x 1 x 0.5 cm³). Transfer fresh human PVAT from DMEM to a 50 mL conical tube containing 25 mL antibiotic solution. Incubate with rocking for 20 min at 4 °C. While the PVAT is in antibiotic solution, thaw an aliquot of dissociation buffer at 37 °C.
2. Add 50 µL of 100x antibiotic/antimycotic solution to 5 mL dissociation buffer and sterilize using a 0.22 µm syringe filter. Add 1 mL of gelatin solution to 1 well of a 24-well plate. In a laminar flow hood, use sterile forceps and scissors to transfer PVAT from the antibiotic solution to a sterile Petri dish. Add 1 mL of pre-warmed dissociation buffer to the tissue and finely mince the entire tissue into a slurry (no pieces larger than ~2 x 2 mm²) using sterile forceps and dissection scissors.
3. Transfer the 1 mL slurry to 4 mL dissociation buffer and incubate the tube on its side in a pre-warmed 37 °C orbital shaker at 200 rpm for 1 h. After 1 h, no visible tissue pieces will be present, and the solution will appear as a cloudy cell suspension.
4. Filter the solution through a 70 µm cell strainer set atop a 50 mL conical tube. Rinse the strainer with an additional 10 mL antibiotic solution to capture as many cells as possible. Do not squeeze the strainer.
5. Pellet the cells for 12 min at 300 x g in a swinging bucket centrifuge.
   NOTE: After centrifugation, the tube will be separated into a fatty top layer of adipocytes, an interphase, and a pellet. The pellet is the stromal vascular fraction containing endothelial cells, immune cells, blood cells, and progenitor cells.
6. Resuspend the pellet in 10 mL of HBSS and centrifuge for 5 min at 300 x g. Repeat this step for a total of 2 washes in HBSS. After the final wash, do not lyse the red blood cells. Repeated attempts with several commercially-available buffers and incubation times have led to marked reductions in progenitor cell attachment and viability.
7. Aspirate gelatin from the 24-well plate. Gently wash the well 1x with HBSS to remove unbound gelatin.
8. Resuspend stromal vascular fraction pellet with intact red blood cells in 1 mL of growth media and seed onto the gelatin-coated well. Add human FGF2 (resuspended in PBS supplemented with 0.01% BSA w/v) to a final concentration of 25 ng/mL in culture medium. Incubate for 24 h at 37 °C with 5% CO₂.
9. On the next day, remove growth media and wash wells 5x with HBSS to remove red blood cells and dead cells. Add in 1 mL of fresh growth media supplemented with 25 ng/mL FGF2.
10. Change the media every 48 h, making sure to supplement with 25 ng/mL fresh FGF2 each time.
11. Cells typically reach 100% confluence 7-10 days after explant; passage cells then:
    1. Aspirate growth media and wash monolayer 2x in 1 mL HBSS. Aspirate all HBSS from the wells and add a few drops of cell dissociation solution.
    2. Tap and swirl the plate several times and incubate at 37 °C with 5% CO₂ for 5–7 min to lift the cells. Add ~1 mL of fresh culture medium to the detached cells and distribute 500 µL to 2 wells of a 24-well plate, each containing 500 µL growth media and 25 ng FGF2.
12. Continue to expand human PVAT-derived cells as indicated in the previous step. Each passage should be no greater than a 1:2 split. Cells are passaged 5–7 times before allocating for differentiation assays.

3. Protocol 2: Culture Human Bone Marrow MSC Colonies

NOTE: Human bone marrow MSC are isolated as described⁸ and stored as early passage frozen stocks in freeze media (70% FBS 20% basal DMEM and 10% DMSO) at ~100,000 cell/mL in liquid N₂.

1. Rapidly thaw a vial of bone marrow MSC from the liquid N₂ in a 37 °C water bath and plate to one well of a 6-well culture plate containing 3 mL of MSC growth media and incubate overnight at 37°C and 5% CO₂.
2. On the next day, aspirate MSC culture media and wash the cells 3x in 2 mL of HBSS. At 100% confluence, aspirate growth media and wash the cells 3x in 2 mL of HBSS. Add 500 µL/well cell detachment solution and incubate at 37 °C and 5% CO₂ for 5 min. Tap the plate to ensure all cells are dislodged and evenly distribute contents to 2 wells of a 6-well plate containing 2 mL MSC growth media.
3. Expand the human bone marrow and PVAT-derived MSC in parallel for approximately 5–7 passages or until sufficient quantities for testing has been achieved.

4. Protocol 3: Plate and Induce Adipogenic, Osteogenic, and Chondrogenic Lineages

1. Plate appropriate numbers of bone marrow and PVAT-derived cells per well of a 12-well plate and appropriate replicates for each experimental condition. For adipogenic and osteogenic conditions, ~200,000–225,000 cells/well are needed, for chondrogenic conditions, 150,000–175,000 cells/well are needed.
   NOTE: We ran a minimum of N = 3 replicate wells for control and induced conditions for each experimental lineage for a total of 2 independent runs (N = 6 replicates total).
2. Disassociate cells from both the human PVAT progenitor cell population and the human bone marrow MSC population using cell detachment solution and incubation at 37 °C and 5% CO₂ for 5 min. Pool populations into separate 15 mL conical vials. Spin the vial down at 500 x g for 7 min to pellet the cells. Resuspend in 1 mL of PBS and use a hemocytometer to estimate the cell number.
3. Plate the cells in 12-well dishes as indicated in step 4.1. Provide separate dishes for induced and non-induced adipogenic, osteogenic, and conditions so that the non-induced condition can be fixed at an earlier time point without disrupting continuing culture of the induced condition.
4. Add 1.5 mL of adipogenic and osteogenic induction media to each well of the induced condition. Add 1.5 mL of adipogenic and osteogenic non-induction media to each well of the non-induced condition. Begin incubation of the adipogenic and osteogenic induced and non-induced cell populations at 37 °C and 5% CO₂.
5. Spin down the remaining volume of human PVAT progenitor cells and human bone marrow MSCs for 7 min at 500 x g.
6. Determine the volume needed to resuspend remaining bone marrow and PVAT-derived cell pellets to achieve a density of 100,000 cells/10 µL (10⁶ cells/mL). Resuspend pellets in the calculated volume of MSC growth media for chondrogenic lineage induction. Gently move the volume of cells up and down using a pipet to ensure a homogenous distribution.
7. Pipette a 10 µL droplet of the concentrated cell solution into the center of each well to form a micromass of 100,000 cells. Place 1 mL of sterile H₂O in the adjacent well to prevent evaporation. Incubate the micromass cultures for 2 h at 37 °C and 5% CO₂ to allow the micromass to aggregate.
8. After 2 hours, carefully add chondrogenic differentiation media spiked with 10 ng/mL human TGFβ1 to each of the induced-condition wells. Carefully add 1.5 mL of the non-induction media (bone marrow MSC growth media) to the non-induced condition wells. Use separate 12-well plates for the induced and non-induced conditions so that the non-induced condition can be fixed at an earlier time point.

5. Protocol 4: Culture Adipogenic, Osteogenic, and Chondrogenic Lineages for 14 Days

1. Culture the induced and non-induced conditions of all three lineages for 4 days at 37 °C and 5% CO₂, refreshing media every 2 days. On day 4, change the induced adipogenic lineage condition from induction media to maintenance media for the remainder of the assay.
2. Fix all of the non-induced conditions in 10% formalin for 12 h. Dispose of formalin and wash all fixed wells 2x in PBS to remove all traces of formalin. Store plates in PBS at 4 °C until processing.
3. Culture the induced conditions for a total of 14 days, refreshing media every 2 days. Add fresh TGFβ1 at 10 ng/mL final concentration with each refresh of the chondrogenic induction media. Continue to culture the adipogenic lineage in the adipogenic maintenance media and osteogenic lineage induction media, refreshing every 2 days. At day 14, fix all induced conditions for 12 h in 10% formalin for staining.
4. Scrape or pour the micromass in the induced chondrogenic condition into a cassette for embedding. Dehydrate the micromass in a series of increasingly concentrated alcohol baths for 5 min, beginning at 70%, then 80%, twice in 95%, and twice more in absolute alcohol. Place the cassette in a dealcoholization agent and then finally embed the cassette in paraffin wax for sectioning and staining.

6. Protocol 5: Staining Adipogenic Condition with Oil Red O

1. Prepare Oil Red O stock solution by dissolving 350 mg of Oil Red O in 100 mL of 100% isopropanol. Mix for 2 h with a stir bar and vacuum through a 0.2 µm filter. Stock solution can be stored in the dark at room temperature for 1 year.
2. Prepare Oil Red O working solution by mixing 3 parts stock solution to 2 parts diH₂0 (e.g. 60 mL of stock solution to 40 mL diH₂0). The final concentration of Oil Red O in the working solution is 2.1 mg/mL.
3. Remove all fluid from wells. Wash each well 2x with 60% isopropanol, making sure to completely remove all water from the sides of the wells. Aspirate 60% isopropanol and quickly add Oil Red O working solution without touching the walls of the wells to cover the bottom. It is critical that this step is done quickly so the wells do not dry.
4. Once the Oil Red O is added, incubate for 10 min at room temperature with gentle rocking.
5. Remove all Oil Red O and immediately add distilled H₂O. Wash with distilled H₂O for 10m to begin to remove unbound Oil Red O. After 10 min, aspirate Oil Red O and repeat the washing procedure 3x.
6. Once washing is complete, add PBS and image the cells. Store stained cells in PBS at 4 °C.

7. Protocol 6: Staining Osteogenic Condition with Alizarin Red

1. Remove all fluid from each well. Add appropriate volume of 2% Alizarin Red stain solution to each well (1.5 mL per well for a 12-well plate) and gently tilt the plate side-to-side until the solution completely covers the bottom of the well.
2. Incubate for 15 min at room temperature. Remove Alizarin Red from the wells. Gently rinse each well four times with distilled H₂O, taking caution to not dislodge calcium crystals. Allow to dry.

8. Protocol 7: Staining Chondrogenic Condition with Masson’s Trichrome

1. Bake slides in a 60 °C dry oven for 30 min. Deparaffinize and hydrate sections to distilled water.
   1. To rehydrate, place slides in three 5 min incubations with dealcoholization agents, followed by 5 min incubations in descending alcohol concentrations as follows: two at 100% (absolute ethanol), two at 95% alcohol, two at 80%, two at 70%, and finally two in distilled H₂O. Slides can remain in H₂O while Bouin’s fixative is prepared.
2. Place 40 mL of Bouin’s fixative in a plastic microwavable coplin jar (uncovered). Microwave on high to bring temp to 55 °C. Place slide in heated solution for 20 min; let stand on the counter covered. Wash in running tap water for 10 min or until all of the yellow color disappears.
3. Stain sections in Weigert’s hematoxylin for 10 min. Wash in running tap water for 10 min.
4. Stain sections in Beibrich’s scarlet acid fuchsin solution for 10 min. Wash 3x 10 min in tap water. Mordant slides in phosphotungstic/phosphomolybdic acid solution for 10 min. Do not rinse.
5. Place slides directly in aniline blue solution for 10 min. Rinse with two brief dips in tap water.
6. Place slides in 1% acetic acid solution for 3 to 5 min. Wash in tap water for 1 min. Place in 95% ethanol for 1 min. Dehydrate as indicated in step 5.4 and mount with synthetic resin.

Results

Isolation of stromal vascular fraction from human PVAT

Figure 1A shows a schematic of the anatomical region where the PVAT overlying the ascending aorta was obtained. We previously described the patient populations undergoing coronary artery bypass grafting from which these samples were derived⁶. Figure 1B shows an example of the...

Discussion

Adipose progenitor cells from different depots vary widely in phenotype and differentiation potential⁹. Culturing PVAT-derived progenitors from a single patient donor in simultaneous induction down three different lineages, adipogenic, osteogenic, and chondrogenic, allows for a well-controlled investigation of the pluripotent capacity of this novel population of progenitor cells. The methodology described in this report can be used to test the differentiation capacity of progenitor cells from huma...

Materials

Name	Company	Catalog Number	Comments
Animal-free collagenase/dispase blend I	Millipore-Sigma	SCR139	50mg
Alcian Blue	NewComerSupply	1003A	1% Aqueous solution pH 2.5
Alizarin Red	Amresco	9436-25G
alpha-MEM	ThermoFisher	12561056
Aniline Blue	NewComerSupply	10073C
Antibiotic/antimycotic	ThermoFisher	15240062
Beibrich's scarlet acid fuchsin	Millipore-Sigma	A3908-25G
b-glycerophosphate	Millipore-Sigma	G9422-10G
Biebrich Scarlet	EKI	2248-25G
Biotin	Millipore-Sigma	B4501-100MG
Bouin's fixative	NewComerSupply	1020A
Bovine serum albumin	Calbiochem	12659	stored at 4 °C
Cell detachment solution	Accutase	AT104
Bell strainer (70 mm)	Corning	352350
Dexamethasone	Millipore-Sigma	D4902-100MG
DMEM	Corning	10-013-CV	4.5 g/L glucose, L-glut and pyruvate
DMEM/F12 medium	ThermoFisher	10565-042	high glucose, glutamax, sodium bicarbinate
DMSO	Millipore-Sigma	D2650
Fetal bovine serum	Atlanta Biologicals	S11550
FGF2	Peprotech	100-18B
Formalin	NewComerSupply	1090
Gelatin, bovine skin	Millipore-Sigma	G9391-500G
Glutamax	ThermoFisher	35050061	glutamine supplement
HBSS	Lonza	10-547F
IBMX	Millipore-Sigma	I5879-250MG
Insulin solution	Millipore-Sigma	I9278-5ML
Oil red O	Millipore-Sigma	O0625-100G
Pantothenic acid	Millipore-Sigma	P5155-100G
Penicillin-streptomycin solution	ThermoFisher	15240062	100ml
Permount	Fisher	SP15-500
Phosphotungstic/phosphomoybdic acid solution	Millipore-Sigma	P4006-100G/221856-100G
Primocin	Invivogen	ant-pm-1	Antimicrobial reagent for culture media.
Rosiglitazone	Millipore-Sigma	R2408-10MG
TGFb1	Peprotech	100-21
Weigert's hematoxylin	EKI	4880-100G